Zhenggang Chen, Shuren Wang, Jinghua Li, Jinhong Han, Qimin Wang, Lei Tong, Wenjun Liu, Fang Yang, Qingyuan Guo, Dawei Guo, Ying Wang
Dept. of Stomatology, Qingdao Municipal Hospital, Qingdao 266071, China;Dept. of Oral and Maxillofacial Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.
Dept. of Stomatology, Jiaozhou People's Hospital, Jiaozhou 266300, China.
Hua Xi Kou Qiang Yi Xue Za Zhi. 2017 Dec 1;35(6):576-582. doi: 10.7518/hxkq.2017.06.003.
RNA interference was used to silence geranylgeranyltransferase Ⅰ(GGTase-Ⅰ) in vitro and to study the effect of GGTase-Ⅰ on the migration and invasion of tongue squamous cancer cells.
Three small interfering RNAs (siRNA) were designed according to the GGTase-Ⅰ sequence by Genebank and were transfected into tongue squamous cancer cells Cal-27 to knock down GGTase-Ⅰ expression. The tested cells were divided into three groups, as follows: the RNA-interfered groups (GGTase-Ⅰ siRNA1, GGTase-Ⅰ siRNA 2, GGTase-Ⅰ siRNA 3), a negative control group (disrupted by random sequence NC-siRNA), and a blank control group. GGTase-Ⅰ and RhoA gene expressions were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The optimum interference group was screened by qRT-PCR and Western blot and was assigned as the experimental group. Matrix metalloproteinase (MMP)-2 and MMP-9 protein expressions were examined by Western blot. GTP-RhoA expression of protein was examined by GST-pull down. The migration and invasion abilities were analyzed by wound healing assay and Transwell motility assay.
GGTase-Ⅰ mRNA and protein expression in Cal-27 decreased significantly after transfection of GGTase-I siRNA (P<0.05). No significant difference of RhoA gene expression was detected. MMP-2, MMP-9, and GTP-RhoA protein expressions decreased significantly (P<0.05). The migration and invasion abilities were inhibited (P<0.05).
To inhibit GGTase-Ⅰ expression, the migration and invasion abilities of tongue squamous cancer cells should also be inhibited. Further studies on GGTase-Ⅰ may provide novel effective molecular targets for tongue squamous cancer cells.
运用RNA干扰技术在体外沉默香叶基香叶基转移酶Ⅰ（GGTase-Ⅰ），研究GGTase-Ⅰ对舌鳞状癌细胞迁移和侵袭能力的影响。
依据Genebank中GGTase-Ⅰ序列设计3条小干扰RNA（siRNA），转染至舌鳞状癌细胞Cal-27以敲低GGTase-Ⅰ表达。将受试细胞分为3组：RNA干扰组（GGTase-Ⅰ siRNA1、GGTase-Ⅰ siRNA 2、GGTase-Ⅰ siRNA 3）、阴性对照组（用随机序列NC-siRNA干扰）和空白对照组。采用定量实时聚合酶链反应（qRT-PCR）及蛋白质印迹法检测GGTase-Ⅰ和RhoA基因表达。通过qRT-PCR和蛋白质印迹法筛选出最佳干扰组作为实验组。采用蛋白质印迹法检测基质金属蛋白酶（MMP）-2和MMP-9蛋白表达。采用GST-pull down法检测蛋白质GTP-RhoA表达。通过划痕愈合实验和Transwell迁移实验分析细胞迁移和侵袭能力。
转染GGTase-I siRNA后，Cal-27细胞中GGTase-Ⅰ mRNA和蛋白表达显著降低（P<0.05）。未检测到RhoA基因表达有显著差异。MMP-2、MMP-9和GTP-RhoA蛋白表达显著降低（P<0.05）。细胞迁移和侵袭能力受到抑制（P<0.05）。
抑制GGTase-Ⅰ表达，舌鳞状癌细胞的迁移和侵袭能力也会受到抑制。对GGTase-Ⅰ的进一步研究可能为舌鳞状癌细胞提供新的有效分子靶点。
